Thermo Fisher Scientific TaqMan Copy Number Assays User guide

Brand: Thermo Fisher Scientific

Model: TaqMan Copy Number Assays

Type: User guide

For Research Use Only. Not for use in diagnostic procedures.

TaqMan™ Copy Number Assays

USER GUIDE

Publication Number 4397425

Revision F

Manufacturer:

Life Technologies Holdings Pte Ltd |

Block 33 |

Marsiling Industrial Estate Road 3 |

#07-06, Singapore 739256

Products:

TaqMan Copy Number Assays

Custom TaqMan Copy Number Assays

Custom Plus TaqMan Copy Number Assays

TaqMan Copy Number Reference Assay, human, RNase P

TaqMan Copy Number Reference Assay, human, TERT

TaqMan Copy Number Reference Assay, mouse, Tfrc

TaqMan Copy Number Reference Assay, mouse, TERT

Manufacturer:

Life Technologies Corporation |

6055 Sunol Blvd |

Pleasanton, CA 94566

Products:

TaqMan Copy Number Assays

Custom TaqMan Copy Number Assays

Custom Plus TaqMan Copy Number Assays

The information in this guide is subject to change without notice.

DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL,

INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT,

INCLUDING YOUR USE OF IT.

Revision history: Pub. No. 4397425

Revision Date Description

F 10 December 2019 Removed troubleshooting and replaced with link to FAQs.

E 6 February 2019 • Added new instruments, Master Mixes, and other applicable

products.

• Added details for dilution of 60X assays.

• Added storage information for gDNA.

• Updated chemistry information.

• Updated order and design information.

• Updated for general style, formatting, and branding.

D September 2010 Baseline for this revision history.

Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept

the terms and conditions of all applicable Limited Use Label Licenses.

Trademarks: All trademarks are the property of Thermo Fisher Scientiﬁc and its subsidiaries unless otherwise speciﬁed. TaqMan is a registered

trademark of Roche Molecular Systems, Inc., used under permission and license. AmpErase is a trademark of Roche Molecular Systems, Inc.

Contents

■CHAPTER 1 Product information ....................................... 5

Product description ............................................................. 5

Assay description ........................................................... 6

Contents and storage ............................................................ 8

TaqMan™ Copy Number Assays ............................................... 8

Reference assay description ................................................. 8

Order copy number assays and reference assays .................................... 9

Master Mixes ................................................................... 9

Required materials not supplied ................................................. 10

Workﬂow ..................................................................... 12

■CHAPTER 2 Guidelines for preparation of gDNA ..................... 13

Guidelines for isolation of high–quality gDNA ...................................... 13

Guidelines to quantify the gDNA ................................................. 13

■CHAPTER 3 Perform PCR ampliﬁcation .............................. 14

Determine the number of sample types and replicates .............................. 14

Dilute the 60X assays ........................................................... 14

Prepare the PCR reactions ...................................................... 14

Prepare the PCR reactions with wet gDNA .................................... 15

Prepare the PCR reactions with dried–down gDNA ............................. 16

Set up and run the real–time PCR instrument ...................................... 17

■CHAPTER 4 Analyze and export results .............................. 19

Analyze the results ............................................................. 19

Export the results .............................................................. 19

Guidelines to export the results ............................................. 20

Import the results to the CopyCaller™ Software ................................... 20

TaqMan

™

Copy Number Assays User Guide

■APPENDIX A Troubleshooting and FAQs .............................. 21

■APPENDIX B Supplemental information .............................. 22

TaqMan™ chemistry ............................................................ 22

About the 5’ nuclease assay ................................................. 22

Best practices for PCR and RT-PCR experiments ................................... 24

Good laboratory practices for PCR and RT-PCR ................................ 24

Use UNG to prevent false-positive ampliﬁcation ............................... 24

Detect ﬂuorescent contaminants ............................................ 25

■APPENDIX C Safety ..................................................... 26

Chemical safety ................................................................ 27

Biological hazard safety ......................................................... 28

■Documentation and support ............................................. 29

Related documentation ......................................................... 29

Customer and technical support ................................................. 30

Limited product warranty ....................................................... 30

Contents

TaqMan

™

Copy Number Assays User Guide

Product information

■Product description .................................................... 5

■Contents and storage .................................................. 8

■Order copy number assays and reference assays ........................... 9

■Master Mixes ......................................................... 9

■Required materials not supplied ........................................ 10

■Workow ........................................................... 12

Product description

Applied Biosystems™ TaqMan™ Copy Number Assays are designed to detect and

measure copy number variation within the human and mouse genomes. Copy

number variation is an important polymorphism associated with genetic diseases

such as cancer, immune diseases, and neurological disorders.

• TaqMan™ Copy Number Assays

– Include an extensive collection of pre–designed assays.

– Are available for genes and known copy number variation regions.

– Are available for human and mouse genomes.

– Provide common vector marker and reporter genes for study of transfected

cells and organisms.

• Custom Plus TaqMan™ Copy Number Assays

– Include bioinformatics analysis on the target and assay sequences.

– Can be designed for other targets of interest in human and mouse genomes.

• Custom TaqMan™ Copy Number Assays

– Can be designed for other targets of interest.

– Do not include bioinformatic analysis.

• TaqMan™ Copy Number Reference Assays

– Are designed to unique human and mouse genomic sequences.

– Are required for relative quantitation of copy number targets.

This document provides instructions for performing and analyzing copy number

variation quantication experiments using the copy number assays and the copy

number reference assays.

TaqMan

™

Copy Number Assays User Guide

The copy number assays are run simultaneously with a copy number reference assay

in a duplex real-time polymerase chain reaction (PCR). The copy number assay

detects the target gene or genomic sequence of interest, and the copy number

reference assay detects a sequence that is known to exist in two copies in a diploid

genome, for example, the human RNase P H1 RNA gene, RPPH1. This method of

relative quantitation is used to determine the relative copy number of the target of

interest in a genomic DNA (gDNA) sample, normalized to the known copy number of

the reference sequence.

The number of copies of the target sequence in each test sample is determined by

relative quantitation (RQ) using the comparative Ct (ΔΔCt) method. This method

measures the Ct dierence (ΔCt) between target and reference sequences, then

compares the ΔCt values of test samples to a calibrator sample known to have two

copies of the target sequence. The copy number of the target is calculated to be two

times the relative quantity.

In a copy number quantication reaction, puried gDNA is combined with the copy

number assay and the copy number reference assay.

The copy number assay includes the following components:

• Two unlabeled primers for amplifying the target sequence of interest.

• One TaqMan™ MGB probe for detecting the target sequence of interest. The probe

incorporates the following items:

– A FAM™ reporter dye, aached to the 5’ end.

– A nonuorescent quencher (NFQ) and a Minor Groove Binder (MGB),

aached to the 3’ end.

Note: MGBs increase the melting temperature (Tm) without increasing probe

length. They allow for the design of shorter probes.

The copy number reference assay includes the following components:

• Two unlabeled primers for amplifying the reference sequence.

• One TaqMan™ TAMRA™ probe for detecting the reference sequence. The probe

incorporates the following items:

– A VIC™ reporter dye, aached to the 5’ end.

– A TAMRA™ quencher, aached to the 3’ end.

Table 1 Description of the reference assays

Assay Purpose Function

Human

TaqMan™ Copy Number Reference

Assay, human, RNase P

Standard reference assay for human

gDNA copy number quantitation

experiments

Detects the Ribonuclease P RNA

component H1 (H1RNA) gene

(RPPH1) on chromosome 14,

cytoband 14q11.2.

TaqMan™ Copy Number Reference

Assay, human, TERT

Alternative reference assay for

human gDNAs

Targets the telomerase reverse

transcriptase (TERT) gene located on

chromosome 5, cytoband 5p15.33.

Assay description

Chapter 1 Product information

Product description

TaqMan

™

Copy Number Assays User Guide

Assay Purpose Function

Mouse

TaqMan™ Copy Number Reference

Assay, mouse, Tfrc

Standard reference assay for mouse

gDNA copy number quantitation

experiments

Detects the transferrin receptor gene

(Tfrc) on chromosome 16, cytoband

16qB3.

TaqMan™ Copy Number Reference

Assay, mouse, TERT

Alternative reference assay for mouse

gDNA

Targets the telomerase reverse

transcriptase (TERT) gene located on

chromosome 13, cytoband 13qC1.

Chapter 1 Product information

Product description

TaqMan

™

Copy Number Assays User Guide

Contents and storage

Each assay contains target-specic forward and reverse primers and FAM™ dye-

labeled MGB probe.

All of the assays are available in 20✕ and 60✕ concentrations and are made to order.

Table 2 TaqMan™ Copy Number Assays

Size

Number of reactions Cat. No.

Shipping and

storage

10–µL reaction 20–µL reaction

TaqMan™

Copy

Number

Assays

Custom

TaqMan™

Copy

Number

Assays

Custom Plus

TaqMan™ Copy

Number Assays

Small (20X) 720 360 4400291 4400294 4442487 Shipped at

ambient

temperature.

Store at −25°C

to −15°C.

Minimize

freeze–thaw

cycles.

Medium

(20X) 1500 750 4400292 4400295 4442520

Large (60X) 5800 2900 4400293 4400296 4442488

Each reference assay detects a single-copy gene in its respective reference genome

assembly. Each reference assay contains reference sequence-specic forward and

reverse primers and a VIC™ dye-labeled TAMRA™ probe.

The reference assays are ordered separately from the copy number assays. For a

description of the reference assays, see Table 1 on page 6.

Table 3 Reference assays

Product

Number of reactions

Cat. No. Shipping and storage

10–µL reactions 20–µL

reactions

Human

TaqMan™ Copy Number

Reference Assay, human, RNase

P, 1 tube (20X)

1500 750 4403326

Shipped at ambient

temperature.

Store at −25°C to −15°C.

Minimize freeze–thaw

cycles.

TaqMan™ Copy Number

Reference Assay, human, RNase

P, 4 tubes (20X)

6000 3000 4403328

TaqMan™ Copy Number

Reference Assay, human, TERT, 1

tube (20X)

1500 750 4403316

TaqMan™ Copy Number

Reference Assay, human, TERT, 4

tubes (20X)

6000 3000 4403315

TaqMan™ Copy

Number Assays

Reference assay

description

Chapter 1 Product information

Contents and storage

TaqMan

™

Copy Number Assays User Guide

Product

Number of reactions

Cat. No. Shipping and storage

10–µL reactions 20–µL

reactions

Mouse

TaqMan™ Copy Number

Reference Assay, mouse, Tfrc, 1

tube (20X)

1500 750 4458366

Shipped at ambient

temperature.

Store at −25°C to −15°C.

Minimize freeze–thaw

cycles.

TaqMan™ Copy Number

Reference Assay, mouse, Tfrc, 4

tubes (20X)

6000 3000 4458367

TaqMan™ Copy Number

Reference Assay, mouse, TERT, 1

tube (20X)

1500 750 4458368

TaqMan™ Copy Number

Reference Assay, mouse, TERT, 4

tubes (20X)

6000 3000 4458369

Order copy number assays and reference assays

• Order TaqMan™ Copy Number Assays.

a. Go to thermosher.com/taqmancnv.

b. Use the Assay Search Tool.

• Design and order Custom TaqMan™ Copy Number Assays and Custom Plus

TaqMan™ Copy Number Assays, and order TaqMan™ Copy Number Reference

Assays.

a. Go to thermosher.com/taqmancustomcnv.

b. Read the background material, then click Order Custom TaqMan™ Copy

Number Assays.

c. Follow the prompts to search for or enter the sequences you need.

Master Mixes

The following Master Mixes are recommended for copy number assays:

• TaqPath™ ProAmp™ Master Mix for samples with PCR inhibitors such as heparin

and hematin

• TaqMan™ Genotyping Master Mix

Chapter 1 Product information

Order copy number assays and reference assays

TaqMan

™

Copy Number Assays User Guide

Table 4 TaqPath™ ProAmp™ Master Mix

Cat. No. Amount Storage

A30865 1 x 1 mL

2–8°C

A30866 1 x 10 mL

A30871 2 x 10 mL

A30867 1 x 50 mL

A30872 2 x 50 mL

Table 5 TaqMan™ Genotyping Master Mix

Cat. No. Amount Storage

4371353 1 x 1 mL

2–8°C

4371355 1 x 10 mL

4381656 2 x 10 mL

4371357 1 x 50 mL

4381657 2 x 50 mL

The following Master Mixes are also compatible with copy number assays:

• TaqMan™ Gene Expression Master Mix

• TaqMan™ Universal PCR Master Mix

• TaqMan™ Universal PCR Master Mix, no AmpErase™ UNG

• TaqMan™ Universal Master Mix II

For details on PCR conditions for the Master Mixes see the appropriate user guide.

Note: TaqMan™ Fast Advanced Master Mix is not recommended for use with copy

number assays.

Required materials not supplied

Unless otherwise indicated, all materials are available through thermosher.com.

MLS: Fisher Scientic (sherscientic.com) or other major laboratory supplier.

Table 6 Recommended products to prepare gDNA

Item Source

Kits to prepare gDNA thermoﬁsher.com/

gdnaprep

Chapter 1 Product information

Required materials not supplied

TaqMan

™

Copy Number Assays User Guide

Table 7 Other materials and equipment required for the workﬂow

Item Source

Real-time PCR instrument, one of the following:

QuantStudio™ 3 or 5 Real-Time PCR System

Contact your local

sales ofﬁce

QuantStudio™ 6 / QuantStudio™ 7 Flex Real-Time PCR System

QuantStudio™ 12K Flex Real–Time PCR System

StepOnePlus™ Real-Time PCR System

ViiA™ 7 Real-Time PCR System

7500/7500 Fast Real-Time PCR System

7900HT Fast Real‑Time PCR System

Software

CopyCaller™ Software (recommended to analyze copy number

assays)

https://

www.thermoﬁsher.co

m/us/en/home/

technical-resources/

software-downloads/

copycaller-

software.html

Equipment

Centrifuge, with adapter for 96-well plates or 384-well plates MLS

Microcentrifuge MLS

Vortex mixer MLS

Pipettes MLS

Tubes, plates, and other consumables

Tubes, plates, and ﬁlm thermoﬁsher.com/

plastics

Aerosol-resistant barrier pipette tips MLS

Disposable gloves MLS

Reagents

Nuclease-free Water AM9930

TaqMan™ DNA Template Reagents 401970

TaqMan™ RNase P Detection Reagents Kit 4316831

(Optional)

TE, pH 8.0 AM9849

Chapter 1 Product information

Required materials not supplied

TaqMan

™

Copy Number Assays User Guide

Workﬂow

Start with gDNA (see page 13)

▼

Determine the number of sample types and replicates (page 14)

▼

Prepare the PCR reactions (page 14)

▼

Set up and run the real–time PCR instrument (page 17)

▼

Analyze and export results (page 19)

Chapter 1 Product information

Workﬂow

TaqMan

™

Copy Number Assays User Guide

Guidelines for preparation of gDNA

Guidelines for isolation of high–quality gDNA

The target template for copy number assays is puried genomic DNA (gDNA). Wet

gDNA or dried-down gDNA can be used. For recommended DNA isolation kits see

Table 6 on page 10.

Guidelines to quantify the gDNA

Quantication of gDNA is recommended. Use one of the following methods:

• The method described in RNase P Quantication for Genotyping Experiments (Pub.

No. MAN0014349 ).

• UV absorbance (A260/A280) measurements. An A260/A280 ratio of greater than 1.7

is recommended for human and mouse gDNA.

The RNase P method is preferred because it is more accurate than UV absorbance,

and it assesses sample quality.

TaqMan

™

Copy Number Assays User Guide

Perform PCR ampliﬁcation

■Determine the number of sample types and replicates ..................... 14

■Dilute the 60X assays ................................................. 14

■Prepare the PCR reactions ............................................. 14

■Set up and run the real–time PCR instrument ............................ 17

Determine the number of sample types and replicates

The following samples are recommended on each plate:

•Samples or Unknowns—gDNA samples in which the copy number of the target

is unknown.

•No–Template Controls (NTC)—A sample that does not contain a DNA template.

It shows the background uorescence and allows for the detection of

contamination.

•Reference sample—A DNA sample with a known copy number for the target of

interest.

Four replicates of each gDNA sample are recommended for reliable copy number

calls.

Dilute the 60X assays

Dilute 60X assays to 20X with TE, pH 8.0. Store the diluted samples at 2–8°C for

immediate use or at −25°C to −15°C for long-term storage. Minimize exposure to light.

Prepare the PCR reactions

Copy number assays can be used with either wet or dried-down DNA. Drying down

the gDNA in plates is the most convenient method for an experiment that requires

multiple plates using the same gDNA or for multiple experiments that use the same

gDNA.

Select the workow to prepare PCR reactions based on whether wet gDNA or dried–

down gDNA is used.

TaqMan

™

Copy Number Assays User Guide

Normalize the wet gDNA sample concentration

Dilute each sample to 5 ng/µL with nuclease–free water or 1✕ TE buer, pH 8.

This is a 5✕ stock solution.

Store the diluted samples at 4°C for immediate use or at −25°C to −15°C for long-term

storage.

Prepare the PCR Reaction Mix

Thaw the copy number assays and the copy number reference assays. Gently vortex

to mix, then centrifuge briey to bring contents to the boom of the tube.

1. Mix the Master Mix thoroughly but gently.

2. In an appropriately-sized centrifuge tube, prepare PCR Reaction Mix according

to the following table.

Component

Volume per well[1]

384–well (0.1–mL) or

96–well fast (0.1–mL)

plate

96–well standard (0.2–

mL) plate

Master Mix (2X) 5.5 µL 11.0 µL

TaqMan™ Copy Number Assay

(20✕)0.55 µL 1.1 µL

TaqMan™ Copy Number

Reference Assay (20✕)0.55 µL 1.1 µL

Nuclease-free Water 2.2 µL 4.4 µL

Total PCR Reaction Mix volume 8.8 µL 17.6 µL

[1] Includes 10% overage.

3. Vortex the tube to mix the PCR Reaction Mix thoroughly, then centrifuge briey

to collect the contents at the boom of the tube.

4. Add the PCR Reaction Mix to the appropriate wells of the plate.

• 384–well (0.1–mL) or 96–well fast (0.1–mL) plate—8 µL per well

• 96–well standard (0.2–mL) plate—16 µL per well

5. Gently vortex the gDNA samples, then add the gDNA to the appropriate wells of

the plate.

• 384–well (0.1–mL) or 96–well fast (0.1–mL) plate—2 µL of gDNA per well

• 96–well standard (0.2–mL) plate—4 µL of gDNA per well

6. Seal the reaction plate with optical adhesive lm, then vortex briey to mix the

contents.

7. Centrifuge the plate briey to collect the contents at the boom of the well.

Proceed to “Set up and run the real–time PCR instrument“ on page 17.

Prepare the PCR

reactions with wet

gDNA

Chapter 3 Perform PCR ampliﬁcation

Prepare the PCR reactions

TaqMan

™

Copy Number Assays User Guide

Dilute and dry the gDNA

The same amout of gDNA must be added to the reaction plate to dry. However,

gDNA samples do not need to be diluted to the same concentration to dry.

1. Transfer the gDNA into each well of a plate.

• 384–well (0.1–mL) plate or 96–well fast (0.1–mL) plate—10 ng per well

• 96–well standard (0.2–mL) plate—20 ng per well

2. Allow the sample to dry at room temperature in an amplicon-free location.

Store the diluted samples at 4°C for immediate use or at −25°C to −15°C for long-term

storage.

Prepare the PCR reactions

Thaw the copy number assays and the copy number reference assays. Gently vortex

to mix, then centrifuge briey to bring contents to the boom of the tube.

1. Mix the Master Mix thoroughly but gently.

2. In an appropriately-sized centrifuge tube, prepare PCR Reaction Mix according

to the following table.

Component

Volume per well[1]

384–well (0.1–mL) or

96–well fast (0.1–mL)

plate

96–well standard (0.2–

mL) plate

Master Mix (2X) 5.5 µL 11.0 µL

TaqMan™ Copy Number Assay

(20✕)0.55 µL 1.1 µL

TaqMan™ Copy Number

Reference Assay (20✕)0.55 µL 1.1 µL

Nuclease-free Water 4.4 µL 8.8 µL

Total PCR Reaction Mix volume 11.0 µL 22.0 µL

[1] Includes 10% overage.

3. Vortex the tube to mix the PCR Reaction Mix thoroughly, then centrifuge briey

to collect the contents at the boom of the tube.

4. Add the PCR Reaction Mix to each well of the reaction plate containing the

dried–down gDNA.

• 384–well (0.1–mL) or 96–well fast (0.1–mL) plate—10 µL per well

• 96–well standard (0.2–mL) plate—20 µL per well

5. Seal the reaction plate with optical adhesive lm, then vortex briey to mix the

contents.

6. Centrifuge the plate briey to collect the contents at the boom of the well.

Proceed to “Set up and run the real–time PCR instrument“ on page 17.

Prepare the PCR

reactions with

dried–down gDNA

Chapter 3 Perform PCR ampliﬁcation

Prepare the PCR reactions

TaqMan

™

Copy Number Assays User Guide

Note: If the plate is not run immediately, protect it from light and store at 2–8°C. If

necessary, the plate can be stored for up to 72 hours if using TaqPath™ ProAmp Master

Mix.

Set up and run the real–time PCR instrument

See the appropriate instrument user guide for detailed instructions to program the

thermal cycling conditions or to run the plate.

Note: The instrument must be congured with the block appropriate for the plate

type.

1. Set the experiment properties.

• Block—384–well or 96–well fast (0.1 mL) or 96–well standard (0.2 mL)

• Experiment type—Standard curve

• Reagents—TaqMan Reagents

• Properties—Standard

2. Dene the targets and the samples, detectors, reporters and quenchers.

Table 8 Required instrument setup information

Instrument Detector

name Target name Reporter Quencher

For copy number assays

7500/7500 Fast Real-Time PCR Systems (software v2.

)

QuantStudio™ 3 and 5 Real-Time PCR Systems

QuantStudio™ 6 and 7 Flex Real-Time PCR Systems

QuantStudio™ 12K Flex Real–Time PCR System

ViiA™ 7 Real-Time PCR System

StepOnePlus™ Real-Time PCR System

NA User–

deﬁned FAM™ dye MGB–NFQ

7500/7500 Fast Real-Time PCR Systems (software

v1.

)[1] FAM™ dye N/A FAM™ dye None

7900HT Fast Real–Time PCR Systems (software v2.

)User–

deﬁned N/A FAM™ dye Nonﬂuorescent

For copy number reference assays

7500/7500 Fast Real-Time PCR Systems (software v2.

)

QuantStudio™ 3 and 5 Real-Time PCR Systems

QuantStudio™ 6 and 7 Flex Real-Time PCR Systems

QuantStudio™ 12K Flex Real–Time PCR System

ViiA™ 7 Real-Time PCR System

StepOnePlus™ Real-Time PCR System

NA User–

deﬁned VIC™ dye TAMRA™ probe

7500/7500 Fast Real-Time PCR Systems (software

v1.

)[1] VIC™ dye NA VIC™ dye TAMRA™ probe

7900HT Fast Real–Time PCR Systems (software v2.

)User–

deﬁned NA VIC™ dye TAMRA™ probe

[1] For SDS Software v1.

, specify FAM™ and VIC™ as the detector names for the copy number assay. SDS Software v1.

does not export dye

information, so the reporter dyes must be specified in the detector name.

Chapter 3 Perform PCR ampliﬁcation

Set up and run the real–time PCR instrument

TaqMan

™

Copy Number Assays User Guide

3. Assign the targets and samples to each well.

4. Set up the thermal protocol.

Note: PCR conditions are the same for TaqPath™ ProAmp™ Master Mix and

TaqMan™ Genotyping Master Mix. TaqPath™ ProAmp™ Master Mix contains

heat–labile UNG, which is active during the reaction setup and is completely

inactivated during the rst ramp to the 95°C hold step.

Step Temperature Time Cycles

Initial denature / enzyme

activation 95°C 10 minutes 1

Denature 95°C 15 seconds 40

Anneal / Extend 60°C 60 seconds

5. Load the plate into the real–time PCR instrument.

6. Start the run.

Chapter 3 Perform PCR ampliﬁcation

Set up and run the real–time PCR instrument

TaqMan

™

Copy Number Assays User Guide

Analyze and export results

■Analyze the results ................................................... 19

■Export the results .................................................... 19

■Import the results to the CopyCaller™ Software .......................... 20

Analyze the results

On the instrument, open the Analysis Seings window and use the following

seings:

• Manual Ct threshold: 0.2

• Autobaseline: On

Apply the seings, close the window, and run the experiment analysis.

For detailed information about data analysis, see the appropriate documentation for

your instrument.

The general guidelines for copy–number data analysis include the following

parameters:

• Ensure that the amplication curves for the copy number reference assay (VIC™

dye signal) in all samples have a distinct linear amplication phase.

• Ensure that the amplication curves for the copy number assay (FAM™ dye

signal) in most wells have a distinct linear amplication phase.

Note: Samples that contain zero copies of the target of interest do not amplify

well, if at all, with the copy number assay. Such samples have high or

undetermined FAM™ Ct values.

• Review any displayed quality check (QC) ags, then review the real-time data of

the associated samples.

Export the results

Export the results les for analysis with the CopyCaller™ Software. For detailed

instructions, see CopyCaller™ Software v2.0 User Guide (Pub. No. 4400042).

Note: If using the ViiA™ 7 Software v1.0 and the CopyCaller™ Software v2.0 for copy

number data analysis, export results using the 7900 Format option. The CopyCaller™

Software cannot open ViiA™ 7 Software v1.0 export les in other formats.

IMPORTANT! Do not modify the exported data les. The CopyCaller™ Software

might not be able to import les that have been modied.

TaqMan

™

Copy Number Assays User Guide

IMPORTANT! If you run multiple copy number assays or copy number reference

assays on each plate, apply distinct assay detector and target names to the document.

See details in “Set up and run the real–time PCR instrument“ on page 17. User-

dened target and detector names allow the CopyCaller™ Software to arrange and

analyze data independently for each assay in a single exported le.

IMPORTANT! If you use a 7500/7500 Fast Real-Time PCR System running SDS

Software v1.X, you must specify FAM™ dye and VIC™ dye as the detector names for

the copy number assays and copy number reference assays, respectively. SDS

Software v1.X does not export dye information, so you must specify the reporter dye

in the detector name.

Assays per

plate

Assay placement

speciﬁed with

separate target

and detector

Procedure

1 — Export the real-time PCR results to a tab-delimited

text (TXT) ﬁle or comma-separated values (CSV) ﬁle.

Yes

Export the real-time PCR results to a single

exported ﬁle (CSV or TXT) that includes all wells of

the plate. The CopyCaller™ Software uses the target

and detector names to distinguish the data from the

different assays.

Export the real-time PCR results of each assay to a

separate exported ﬁle.

1. Select the wells of the plate that contain the

data from one of the copy number assays.

2. Select File4Export, then export the data from

the selected wells to a data ﬁle.

3. Repeat to export the data from the other

assays present on the plate.

Note: To help with organization, name each

exported ﬁle according to the assay data it contains.

Import the results to the CopyCaller™ Software

For more information, see the CopyCaller™ Software v2.0 User Guide (Pub. No. 4400042).

1. Import the exported real-time PCR results le into the software.

2. Run the analysis to determine the copy number for your target in each sample.

Guidelines to

export the results

Chapter 4 Analyze and export results

Import the results to the CopyCaller

™

Software

TaqMan

™

Copy Number Assays User Guide

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Thermo Fisher Scientific TaqMan Copy Number Assays User guide

Brand: Thermo Fisher Scientific

Model: TaqMan Copy Number Assays

Type: User guide

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Thermo Fisher Scientific TaqMan Copy Number Assays User guide

Thermo Fisher Scientific TaqMan Copy Number Assays User guide

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